Non-isotopic in situ hybridization is a powerful molecular approach for detecting and localizing specific nucleic acid sequences within interphase nuclei or chromosomes. Single viral copies were detected by fluorescent signals after hybridization with biotinylated virus DNA probes. This has a practical significance for detecting DNA and RNA viruses in malignant and premalignant lesions. On a cervical carcinoma cell line (C4-1), human papillomavirus-18 (HPV-18) sequences were localized at region 8q2l on an 8;12 rearranged chromosome. In a Burkitt's lymphoma cell line, Epstein-Barr virus sequences were mapped on chromosome 2pl3 adjacent to a viral modification site. In both lines viral integration corresponded with the location of a fragile site. A nontumorigenic line (CX16-2) derived from exocervical epithelial cells transfected with recombinant HPV-16, hoarbor viral sequences on chromosome 2 near ets-2 gene. The ets-2 specific m-RNA level was elevated in the absence of structural gene alterations. However, no linkage between ets-2 and HPV-16 sequences was established by pulse field gel electrophoresis using several rarecutting restriction enzymes. Thus, HPV sequences can influence, from a distance, proto-oncogene expression. Several HPV-16 integration sites exhibited an aberrant late replication pattern. Incomplete chromatin condensation and recombination are consequences of the replication junction that flank late replicating DNA and can explain the origin of chromosomal changes associated with the cell's immortality. Neu proto-oncogene, the rat homolog of the human erb-B-2-gene, was neither amplified nor overexpressed in two rat mammary cell lines neoplastically transformed in vitro by a chemical carcinogen. A cDNA for the gene that encodes a human cytosolic thyroid hormone binding protein (p58) was mapped by in situ hybridization to 15q24-25. This localization may serve as a useful marker for Tay-Sachs disease and will permit assessment of the effect chromosome alterations involving this region have on human malignancies.